Septicemia, which is the presence of pathogenic microorganisms in the blood, is one of the most serious types of infection encountered in modern medicine. Despite the armament of antimicrobial agents available today, the mortality rate for septicemia is approximately twenty-five percent, and when accompanied with septic shock, the mortality rate increases to about sixty percent. Patients suffering from debilitating diseases, undergoing major surgery, or receiving immunosuppressive drug therapy are especially prone to septicemia. Accurate, rapid quantitative analysis of blood specimens from patients suspected of septicemia is recognized as one of the most critical functions of clinical microbiology.
Transfusion of contaminated blood or blood products also represents a serious medical problem, often resulting in life-threatening septicemic shock or even death of the recipient. These risks are common for both autologous transfusions, where the blood donor is also the recipient, and homologous transfusions, where the blood donor is someone other than the recipient. Consequently, bacterial contamination of blood collected and processed by blood banks for transfusion represents a major threat to national blood supply and the national health.
The microorganisms associated with transfusion sepsis have certain characteristics. These organisms are capable of persisting in the blood for an extended period of time and often in high titers. It has been previously reported that introduction of a single organism into a platelet product can result in a final concentration of 10.sup.4 to 10.sup.9 colony forming units per milliliter (cfu/ml) of blood within three days at room temperature. (Braine, H., et al. 1986. Bacterial sepsis secondary to platelet transfusion: an adverse effect of extended storage at room temperature. Transfusion 26:391-393) They may exhibit a latent carrier state allowing an individual to be infected without manifesting any symptoms, or they may cause only subclinical infections. They are also stable in cold, stored blood. Among the microorganisms reported as being associated with transfusion sepsis are Yersinia enterocolitica, Pseudomonas sp., Achromobacter sp., Escherichia coli, Salmonella sp., Staphylococcus epidermidis, Staphylococcus aureus, Flavobacterium sp., and Streptococcus viridans. (Ulrich, P. and G. Vyas. 1992. Blood-borne infections associated with transfusion. J. Intensive Care Med. 7:67-83)
Identifying probable causes as well as possible solutions for transfusion sepsis requires knowledge of blood collection, process and storage. Whole blood obtained from "healthy" blood donors is routinely processed by blood banks to form various blood products which are subsequently transfused to patients for various purposes. A major source of possible donor blood contamination involves the collection of blood from an individual suffering from infection. During the infection process, microorganisms are seeded from the sight of infection into the bloodstream from which they are subsequently collected along with the donated blood. In an effort to prevent transfusion sepsis caused by transfusing blood collected from an individual suffering from infection, the donor's medical history is reviewed for situations which might signify an increased risk of harboring an infection, including exposure to infectious diseases by traveling to endemic areas; invasive procedures such as surgery, or social behavior such as homosexuality or drug addiction which are associated with increased risk of exposure to infectious diseases; or immunization against particular infectious agents. The donor is also given a physical examination which includes measuring the donor's body temperature as an indicator of possible infection. (Technical Manual. 1990. Edited by R. H. Walker, 10th ed., American Association of Blood Banks, Arlington, Va., pp. 3-8) Despite these attempts to screen infected donors, blood banks cannot adequately prevent blood collection from a donor who is dishonest when answering medical history questions or who unknowingly has a subclinical infection.
In a typical example of blood collection, approximately 450 milliliters of whole blood is collected from the donor by inserting a phlebotomy needle into a large vein in the donor's arm. The phlebotomy needle is connected to the delivery tubing leading into the primary blood collection bag. The needle insertion process also represents a major source of potential contamination. Despite procedures to disinfect the donor's arm with antimicrobial agents prior to phlebotomy, bacteria from the donor's skin, hair follicles, or sebaceous glands can enter a blood bag in the skin "core" cut out by the venipuncture needle. (Morrow, J., et al. 1991. Septic reactions to platelet transfusions. JAMA 266:555-558)
The primary bag contains an anticoagulant (e.g., citrate phosphate dextrose solution (CPD)) to prevent clotting, and during the collection process, the primary bag is inverted by a phlebotomist to disperse the anticoagulant evenly throughout the blood. Shortly after collection, the blood in the delivery tubing is "stripped" into the primary bag. After inverting the primary bag several times, the delivery tubing is allowed to refill with the anticoagulated blood. The delivery tubing is then heat-sealed into several detachable segments, providing samples of the collected blood for subsequent testing.
In blood processing, the whole blood donation is separated into various blood components. Historically, this process was performed in an "open" system where the blood was exposed to the environment during each separation procedure, and with exposure, the opportunity for contamination existed. In modern blood banking, blood processing is routinely performed using a closed blood bag system, where the primary bag has integrally attached satellite bags into which blood can be split into various blood products and subsequently stored without breaking hermetic seals, preventing exposure of the blood to the environment and possible contamination.
The primary bag of whole blood is first centrifuged at about 2100 rpm for about six minutes, causing the heavier red blood cells (RBCs) and about 1.times.10.sup.9 white blood cells (WBCs) to sediment. The platelet-rich plasma, usually containing low numbers of RBCs and about 4.times.10.sup.7 WBCs, is then expressed through connecting tubing from the primary bag into a satellite bag. The RBC sediment remaining in the primary bag contains approximately 210 milliliters (ml) RBCs, residual plasma, and anticoagulant. A red cell preservative solution (e.g., Adsol containing dextrose, sodium chloride, mannitol, and adenine) is added to the RBCs, producing approximately 300 ml packed red cells which currently have a shelf life of 42 days when stored at 6.degree. C.
The satellite bag containing approximately 260 ml platelet-rich plasma and 60 ml anticoagulant may be centrifuged at about 3800 rpm for about three minutes, separating the platelets and from the plasma. The residual plasma and anticoagulant is expressed through connecting tubing into another satellite bag. The platelet concentrate containing approximately 45 ml platelet concentrate and 10 ml anticoagulant is then stored at 1.degree.-6.degree. C. for up to three days or on a rotator at 20.degree.-24.degree. C. for up to five days.
The plasma may be frozen within eight hours to produce fresh frozen plasma with a shelf life of one year when stored at -18.degree. C. Alternately, it may be frozen, thawed, and then centrifuged at approximately 4200 rpm for five minutes to separate plasma (non-transfusable liquid recovered plasma) from cryoprecipitate which has a shelf life of one year when stored at -18.degree. C. (Technical Manual. 1990. Edited by R. H. Walker, 10th ed., American Association of Blood Banks, Arlington, Va., pp. 43-55, 634-641)
Hemapheresis is another blood collection methodology which is becoming more prevalent in the blood banking industry. There are two categories of hemapheresis: 1) plasmapheresis for the collection of plasma and 2) cytapheresis for the collection of cellular blood components such as platelets and granulocytes. In these procedures, whole blood is removed from a donor; the blood is separated into components; the desired component is retained; and the remaining blood elements are recombined and returned to the donor. Cell separators utilize centrifugal force and the different densities of various blood components to achieve separation of the desired component. Platelet concentrates obtained by apheresis with a closed-system cell separator are stored at 1.degree.-6.degree. C. for up to three days or on a rotator at 20.degree.-24.degree. C. for up to five days; granulocyte concentrates, up to 24 hours at 20.degree.-24.degree. C. Many blood banks perform apheresis for the production of platelets and granulocytes preferentially over production from whole blood donations for a variety of reasons including: 1) the procedure can be performed more often than whole blood donation (once every forty-eight hours as opposed to once every fifty-six days) and 2) plateletpheresis typically yields a platelet unit volume equivalent to a pooled platelet unit made by combining seven to ten single platelet units obtained by processing whole blood donations. Clinicians in transfusion medicine report a reduction in serious antigen-antibody reactions due to the fact that the recipient is exposed to the antigenic properties of one person as opposed to the combined antigenic properties of multiple donors for a given transfusion. (Technical Manual. 1990. Edited by R. H. Walker, 10th ed., American Association of Blood Banks, Arlington, Va., pp. 19-24)
In apheresis procedures, the potential of contamination from infected donors and from the venipuncture needle "core" still exists. Most cytapheresis procedures utilize two venipunctures, inserting one phlebotomy needle into a vein of one arm for the removal of whole blood and another needle into a vein of the other arm for return of the remaining blood elements. This method of double phlebotomy increases the chances of contaminating the blood product.
Definitive steps have been taken in blood collection, processing and storage to reduce the possibility of bacterial contamination and subsequent transfusion sepsis. As mentioned previously, screening of blood donors in an attempt to prevent an individual suffering from an infection from donating blood, scrubbing regimens for cleaning the surface of the donor's arm, and the use of closed blood bag systems have decreased the risk of contaminated blood or blood products. Yet, despite these attempts to prevent or minimize contamination, transfusion sepsis continues to threaten the recipients of contaminated blood or blood products.
At a special session of the FDA's Blood Products Advisory Board on Yersinia contamination in platelets held in Washington, D.C. in May, 1991, several suggestions were presented as solutions to blood/blood product contamination: 1) attempting to improve the arm preparation prior to venipuncture, 2) adding antibiotics to the blood products, 3) shortening the storage time, 4) lowering storage temperatures, 5) holding blood at room temperature for several hours prior to processing and storage, 6) filtration, and 7) bacterial detection systems. It was concluded that surface disinfection of the arm does not eliminate contamination from the venipuncture needle "core" and improvements in arm preparation would not significantly lower the risk of transfusion sepsis. There was some skepticism concerning the effects of long term storage of antibiotics in the blood or blood product and its ultimate effect upon the transfusion recipient; therefore, the FDA Advisory Board did not consider applying this methodology to the problem of transfusion sepsis.
Another resolution suggested for the blood contamination crisis is to shorten the storage time allowed for blood and blood products. With the advent of closed blood bag systems and increased blood cell viability due to improvements in plastics chemistry, the storage time for certain blood products from the time of collection to the time of transfusion has been increased (RBC concentrates, from twenty-one to forty-two days; platelet products at room temperature, from three to seven days). Investigation of documented cases of transfusion sepsis have shown that the longer the storage time, the greater the risk of life-threatening transfusion sepsis. Severe medical complications resulting from the infusion of contaminated blood products has generally been associated with refrigerated units stored in excess of twenty-five days, and units stored at room temperature, five to seven days. Thus, it has been suggested that reducing the storage time for a blood product would lower its bioburden to "safe levels" for transfusion. However, shortening the allowable storage time for blood products does not totally eliminate the risk. Further, shortening storage times creates a serious imbalance between the public's blood requirements and available blood supply, producing a major negative impact on the cost effectiveness of blood centers and endangering the national blood supply.
Storing collected whole blood and most blood products at refrigeration temperature has also been suggested as a means of either preventing or reducing the growth of any bacteria introduced either from the donor blood or during the collection process. However, despite refrigeration of blood products, certain microorganisms routinely found in contaminated blood are capable of growing and reproducing from 10.sup.2 cfu/ml to concentrations of 10.sup.8 to 10.sup.9 cfu/ml when stored at refrigeration temperatures for 15 to 30 days.
The suggestion that whole blood be held at about 22.degree. for 4 to 20 hours in order to exploit the natural self-sterilizing properties of blood as a method of improving the safety of the blood supply also fails to solve the problem. Blood has been reported to contain two basic types of immunity: (1) an innate, nonspecific immune mechanism consisting of phagocytes such as granulocytes and monocytes, and humoral components such as complement proteins, and (2) a learned, specific immune mechanism involving the antibody reactions of lymphocytes and immunoglobulins to antimicrobial antigens. At the time of collection, blood collected from a healthy, immunocompetent donor contains a significant number of phagocytes: approximately 3.times.10.sup.9 WBCs, consisting of granulocytes (34%-83%), monocytes (3%-15%), and lymphocytes (12%-50%). (Merck Manual. 1992. 16th ed. (R. Berkow, ed.), Merck & Co, Rathway, N.J., p. 1141) While it is suggested that granulocytes will disintegrate within 1-5 days, monocytes are known to survive for over twenty days. Therefore, this approach to handling the blood supply relies on the donor being immunocompetent at the time of collection. Further, the immune response to the presence of microorganisms in the blood is first directed toward phagocytosis, which is facilitated by opsonization, or coating of a microorganism with antibody and/or complement proteins to promote its adherence and ingestion. Therefore, an effective immune response depends upon a successful antibody system, the activation of the complement cascade system and other humoral factors, and a intact phagocytic system. In practice, the attempt to exploit blood's self-sterilizing properties is ineffective because it is dependent on individual donor immunocompetence and it is thwarted by recommended storage conditions. Certain microorganisms such as Yersinia enterocolitica thrive at 4.degree.-6.degree. C., routine storage temperatures for stored blood. It is reported that humoral factors such as complement are inactivated and phagocytosis is reduced by about 70% at refrigeration temperatures. Moreover, it has been reported that phagocytized bacteria can only be killed by an oxidative burst which can occur only at higher temperatures such as 22.degree.-37.degree. C. (Pietersz, Proceedings from the 36th Meeting of the FDA Blood Products Advisory Committee, (1991)) Yet, to increase the viability of the RBCs, whole blood is routinely stored at 1.degree.-10.degree. C. prior to processing, if platelets are not to be prepared from the unit. After processing, many blood products are stored at 1.degree.-6.degree. C. Therefore, the self-sterilizing properties of the blood are blocked by recommended storage conditions.
The FDA Advisory Board concluded that research should be directed into the areas of leukocyte filtration and the development of bacterial detection systems for screening blood bank products for contamination. Leukocyte filtration devices have been developed for the production of leukopoor RBCs where WBCs, responsible for phagocytosis of microorganisms, are removed from the blood prior to processing in an effort to remove infectious agents and to reduce nonhemolytic transfusion reactions (e.g., graft versus host disease). (Technical Manual. 1990. Edited by R. H. Walker, 10th ed., American Association of Blood Banks, Arlington, Va.) (Ulrich, P. and G. Vyas. 1992. Blood-borne infections associated with transfusion. J. Intensive Care Med. 7:67-83)
In a study of various leukocyte filtration devices for the production of leukocyte-poor blood and blood products, the number of WBCs per packed RBC unit was reportedly reduced approximately 89% to 99%, or from about 4.times.10.sup.9 WBCs/unit to about 10.sup.7 -10.sup.9 WBCs/unit. (Rawal, B., et al. 1990. Dual reduction in the immunologic and infectious complications of transfusion by filtration/removal of leukocytes from donor blood soon after collection. Transfusion Medicine Reviews IV:36-41) However, WBCs remaining in such filtered blood may contain microorganisms either phagocytized by or attached to the remaining WBCs. Subsequent rupturing of WBCs, either through natural death of the phagocyte over time or by mechanical disruption upon processing, will cause microorganisms to be released to contaminate the blood. Further, microorganisms can remain unassociated with WBCs, and filtration devices have not been shown to clear unattached bacteria from blood products. It is disclosed herein that certain bacteria may be associated with RBCs, and consequently, would not be removed from packed RBCs by a leukocyte filtering device.
Development of a rapid, sensitive, reliable, cost-effective test procedure which will identify contaminated blood products prior to transfusion has also been proposed. Suggested procedures for screening blood products include gram staining, acridine orange staining, endotoxin assay, and microbial cultural analysis. Gram staining requires a high concentration of bacteria of at least 10.sup.5 -10.sup.6 cfu/ml, and visualization of the bacteria is difficult due to the blood cells and bacteria staining the same color. Although acridine orange staining is more easily visualized with the blood cells staining pale green and the bacteria staining bright fluorescent orange, it is less sensitive, requiring greater than 10.sup.5 -10.sup.6 cfu/ml for detection. As a fluorescent staining technique, it is also susceptible to a high false positive/false negative rate. The endotoxin assay is a relatively expensive technique with a high false positive rate, requiring approximately 10.sup.5 cfu/ml. This procedure also fails to detect certain classes of bacteria which have been associated with transfusion sepsis (e.g., Staphylococcus species).
Another approach to identifying contaminated blood is conventional microbial cultural analysis. One method routinely used to detect septicemia in patients is the broth-based blood culture bottle system which is designed to encourage all microorganisms in a blood sample to replicate to a detectable level, thus providing qualitative-only microbial cultural analysis. A routine blood culture bottle contains a nutrient broth, e.g., tryptic soy broth, brain heart infusion, supplemented peptone, or thioglycolate broth, and an anticoagulant such as sodium polyanethol sulfonate (SPS) (Difco Laboratories, Detroit, Mich.) (BBL Microbiology Systems, Cockeysville, Md.). SPS is most commonly used in broth blood culture media at a concentration of 0.025%-0.03%. As an anticoagulant, SPS prevents clotting of the blood which would entrap the bacteria and prevent their detection. SPS is also reported to act as an anticomplementary and antiphagocytic agent, and to interfere with the antimicrobial action of aminoglycosides, providing further protection for the bacteria in the broth blood culture. In general, about 5 ml of blood is added to a blood culture bottle containing 45 ml of broth, representing a substantial dilution (1:10). This dilution, coupled with 0.03% SPS, generally provides adequate protection against complement and phagocytosis for the microorganisms to grow in the broth culture. The inoculated bottle is incubated at 35.degree. C. and after twenty-four and forty-eight hours, a 0.1-0.5 ml aliquot of the blood culture is manually subcultured (i.e., blind subculture) onto routine nutritional agar medium (about 25 ml agar medium per 110.times.15 mm plate; hereinafter referred-to as "agar medium") such as blood agar or chocolate agar plates. The plates are incubated at 35.degree. C. and are examined for microbial growth after twenty-four to forty-eight hours. The blood culture bottle is checked visually or growth/no growth detected via automated systems on a daily basis for up to seven to fourteen days for any evidence of microbial growth such as turbidity, hemolysis, gas production, or formation of discrete colonies. If visible growth is detected, the blood cultures are subcultured onto isolation media. At routine intervals, blind subcultures are performed. (Manual of Clinical Microbiology, 1985. Edited by E. H. Lennette, 4th ed., American Association for Microbiology, Washington, D.C., pp. 75-76) (Finegold, S. M. and E. J. Baron. 1986. Bailey and Scott's Diagnostic Microbiology, 7th ed., C. V. Mosby Co., St. Louis, Mo., p. 217-218)
Basic broth blood culture methodology may require several days of incubation/subculture in order to obtain a pure isolate of a bacterium and yield minimal information (i.e., growth vs. no growth). If more than one organism is present in a patient blood sample, the more fastidious or slow growing organism is usually overgrown by fast-growing organisms and consequently is not detected. This methodology does not distinguish between "real" contamination in a blood sample versus laboratory contamination, i.e., contamination inadvertently introduced into the test system from the environment or by personnel during cultural analysis. Multiple broth blood cultures on one patient are usually performed, utilizing repetitive culturing of the same organism to distinguish between real and laboratory contamination. If broth-based methodologies are adopted for bacterial detection in blood bank products, any bacterial growth in the cultural analysis results in an automatic disposal of the blood product. Between 1% to 6% of all samples analyzed will yield inaccurate results due to laboratory contamination, representing a significant loss of blood product to the blood banking industry.
Another method of clinical microbial cultural analysis is the lysis-centrifugation procedure taught by Dorn et al. in U.S. Pat. No. 4,164,449 issued Aug. 14, 1979, and entitled "Surface Separation Technique for The Detection of Microbial Pathogens," and sold commercially as the ISOLATOR.TM.10 (Carter-Wallace, Inc., Cranbury, N.J. 08512-0181) which contains 0.7 milliliters of an aqueous reagent containing 8 milliliters/liter polypropylene glycol (P-2000), 9.6 grams/liter sodium polyanethol sulfonate, 1 unit activity purified saponin,and 16 grams/liter ethylene diaminetetraacetic acid. In this procedure, patient blood is collected into a blood culture centrifugation tube which contains a chemical cocktail comprising a lysing agent of purified saponin (purified according to the procedure taught by Dorn in U.S. Pat. No. 3,883,425 issued May 13, 1975 and entitled "Detoxification of Saponins"; hereinafter referred to as "purified saponin" or "saponin", and hereby incorporated herein by reference), SPS as an anticoagulant, and ethylenediaminetetraacetic acid (EDTA) as a calcium chelator. In lysis-centrifugation methodology, about 10 ml of blood is collected into a blood culture centrifugation tube, and the tube is inverted several times to facilitate mixing of the sample with the cocktail and subsequent lysing of the blood. The lysed blood sample is then centrifuged, the supernatant discarded, and the sediment which contains the bacteria plated at about 0.3-0.5 ml per plate onto appropriate isolation solid agar media such as blood agar and chocolate agar. Within twenty-four to forty-eight hours, organisms cultured from the blood appear as discrete colonies on the isolation agar media. A lysis-only pediatric version covered by U.S. Pat. No. 4,164,449 described above and sold commercially as the ISOLATOR.TM.1.5 (Carter-Wallace, Inc.) which contains 0.1 milliliters of an aqueous reagent containing 8 milliliters/liter polypropylene (P-2000), 9.6 grams/liter sodium polyanethol sulfonate, and 1 unit activity purified saponin, is also available to which 1.5 ml of blood is added to a formulation containing SPS and purified saponin as a lysing agent, and about 0.5 ml of the lysed blood is plated directly onto appropriate isolation solid agar media.
Certain aspects of the ISOLATOR.TM.10 and ISOLATOR.TM.1.5 methodologies are reported to be advantageous for clinical microbial analysis and would be applicable to blood banking. The presence of 0.06% SPS in the ISOLATOR.TM.10 and ISOLATOR.TM.1.5 is reported to inhibit the phagocytic activity of granulocytes and monocytes, and the normal antimicrobial activity of serum. (Finegold, S. M. and E. J. Baron. 1986. Bailey and Scott's Diagnostic Microbiology, 7th ed., C. V. Mosby Co., St. Louis, Mo., p. 221-222) Further, the lysis-centrifugation and lysis-only methodologies generally require twenty-four hours to not only obtain a pure isolate of a bacterium, but also to quantitate the number of bacteria per milliliter of blood. Using the quantitative aspect of the methodology, it would be possible to distinguish between real contamination in the blood product versus laboratory contamination (generally in the range of .ltoreq.10 cfu/ml) if the bacterial count in the contaminated blood product is high, i.e., .gtoreq.10.sup.2 cfu/ml.
Although the problem of microbial contamination of blood has been recognized and various solutions attempted, there remains a need to identify potentially unsafe blood in a rapid and effective manner in order to provide for an effective balance between safety of the blood supply and having sufficient blood and blood products available to meet the needs of patients requiring transfusions.
A new method of quantitative microbial cultural analysis has now been found which results in a substantial improvement in microbial detection over the systems described above. By using this method to culture blood bank products held beyond "safe" periods (e.g., packed RBCs, .gtoreq.10 days; platelets, .gtoreq.3 days), it is possible to distinguish real blood product contamination from laboratory contamination. This methodology can also be utilized in improving quantitative blood cultures of septicemic patients. It is also applicable to the microbiological cultural analysis of veterinary specimens and of blood containing food products such as those encountered in the meat, poultry and seafood industries.